LAUSR

Amyloid β (Aβ) peptide contributes to Alzheimer's disease (AD) by a yet unidentified mechanism. In brain tissue, Aβ occurs in various forms, including a undecapeptide Aβ(25-35) (GSNKGAIIGLM), which exerts neurotoxic effect through mitochondrial dysfunction and/or Ca2+-permeable pore formation in cell membranes. This work was aimed at biophysical characterization of pores formed by Aβ(25-35) in membranes of unilamellar vesicles composed of zwitterionic POPC, acidic POPG, and varying fractions of cholesterol. Vesicles were made by extrusion, in a buffer containing 6 mM Quin-2, a Ca2+-dependent fluorophore. External Quin-2 was removed using a Sephadex G-50 column, and 6 mM CaCl2 was added externally to the vesicles. Aβ(25-35) was incubated in an aqueous buffer for 3 hours to form oligomers, and was added to the vesicles, resulting in gradual increase in Quin-2 fluorescence, interpreted in terms of membrane pore formation by the peptide, Ca2+ influx and binding to intravesicular Quin-2. The positive and negative controls involved addition of a non-fluorescent calcium ionophore Br-A23187 or blank buffer, respectively. The pore forming activity of Aβ(25-35) was dependent on the lipid composition of the vesicles and the ionic strength of the buffer. High ionic strengths (150 mM NaCl and above) significantly suppress pore formation, indicating the importance of electrostatic interactions between the cationic peptide (Lys28) and anionic membranes. CD spectra of Aβ(25-35) in same vesicle samples displayed a minimum around 210 nm and a shoulder at 222 nm, whereas the peptide in a buffer without lipid showed a minimum around 206 nm, indicating membrane induced α-helix formation. The effect of membrane cholesterol on Aβ(25-35) pore formation is underway and may elucidate the role of cholesterol in AD pathogenesis. Combined with FTIR analysis, these studies will provide the structure and function of membrane pores formed by Aβ(25-35).